Multiple Functionalities of Reduced Flavin in the Non-Redox Reaction

Archives of Biochemistry and Biophysics 632 (2017) 59e65

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Archives of Biochemistry and Biophysics

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Review article Multiple functionalities of reduced ﬂavin in the non-redox reaction catalyzed by UDP-galactopyranose mutase

* ** Pablo Sobrado a, , John J. Tanner b, a Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA b Departments of Biochemistry and Chemistry, University of Missouri, Columbia, MO 65211, USA article info abstract

Article history: Flavin cofactors are widely used by enzymes to catalyze a broad range of chemical reactions. Tradi- Received 2 June 2017 tionally, flavins in enzymes are regarded as redox centers, which enable enzymes to catalyze the Received in revised form oxidation or reduction of substrates. However, a new class of flavoenzyme has emerged over the past 21 June 2017 quarter century in which the flavin functions as a catalytic center in a non-redox reaction. Here we Accepted 22 June 2017 introduce the unifying concept of flavin hot spots to understand and categorize the mechanisms and Available online 24 June 2017 reactivities of both traditional and noncanonical flavoenzymes. The major hot spots of reactivity include the N5, C4a, and C4O atoms of the isoalloxazine, and the 20 hydroxyl of the ribityl chain. The role of hot Keywords: fl fl Flavin-dependent reaction spots in traditional avoenzymes, such as monooxygenases, is brie y reviewed. A more detailed fl fl Flavin adducts description of avin hot spots in noncanonical avoenzymes is provided, with a focus on UDP- Galactofuranose galactopyranose mutase, where the N5 functions as a nucleophile that attacks the anomeric carbon Non-redox reaction atom of the substrate. Recent results from mechanistic enzymology, kinetic crystallography, and Neglected diseases computational chemistry provide a complete picture of the chemical mechanism of UDP- Tuberculosis galactopyranose mutase. Redox-switch © 2017 Elsevier Inc. All rights reserved. Conformational changes Protein dynamics

Contents

1. Introduction ...... 59 2. Formation of flavin N5-adducts ...... 60 3. Priming the flavin-N5 for reactivity in non-redox reactions ...... 61 4. Activation of intermediates in catalysis: role of the flavin-C4O in proton transfer ...... 62 5. Summary and outlook ...... 62 Acknowledgments ...... 64 References...... 64

1. Introduction leverage amino acid functional groups to catalyze chemical reactions [1]. Three-dimensional constellations of amino acids opti- Enzymes exhibit extraordinary catalytic power and diversity, mized by evolution are capable of catalyzing diverse reactions, which remains unmatched by man-made catalysts. All enzymes including phosphoryl transfer, peptide bond hydrolysis, and isomerization reactions among many others [2e4]. Cofactors expand the chemical repertoire of enzymes, allowing more complex chemical * Corresponding author. reactions to occur. For example, heme-dependent enzymes can ** Corresponding author. cleave C-H bonds via the formation of Fe-oxo species [5]. Pyridoxal E-mail addresses: [email protected] (P. Sobrado), [email protected] phosphate-dependent enzymes form a covalent aldimine (J.J. Tanner). http://dx.doi.org/10.1016/j.abb.2017.06.015 0003-9861/© 2017 Elsevier Inc. All rights reserved. 60 P. Sobrado, J.J. Tanner / Archives of Biochemistry and Biophysics 632 (2017) 59e65 intermediate that facilitates a-C-H bond cleavage, which is impor- 2. Formation of flavin N5-adducts tant in racemization and elimination reactions [6]. Numerous oxi- doreductases use nicotinamide adenine dinucleotide cofactors in The reactivity of the N5-atom of the isoalloxazine ring was hydride transfer reactions. Photosynthetic reaction centers perhaps originally investigated in model compounds where the spectral and represent the pinnacle of cofactor-assisted catalysis [7]. fluorescent properties of N5-alkylated flavins were reported [17,18]. Flavin cofactors are also widely used by enzymes to catalyze a The presence of N5-flavin adducts in enzymes was demonstrated in broad range of chemical reactions. Flavins are derived from ribo- the reaction of D-amino acid oxidase with a nitroethane anion flavin (vitamin B2) and are processed into flavin mononucleotide (Scheme 2) in the presence of cyanide and lactate oxidase with (FMN) or flavin adenine dinucleotide (FAD) in the cell [8]. Typically, glycolate [19]. It was later demonstrated that an N5-adduct played a flavoenzymes catalyze oxidation-reduction reactions, where the role in the catalytic cycle of nitroalkane oxidase [20]. This adduct flavin cycles between the oxidized and reduced states, such as in was later trapped with cyanide and the structure elucidated by x- hydride transfer reactions. In other redox reactions, flavoenzymes ray crystallography [21]. N5-adducts have also been proposed in form a transient semiquinone, such as in electron transfer reactions the reaction of monoamine oxidase with several substrates and [8e12]. inhibitors [22]. The b-Cl-elimination activity of D-amino acid oxi- The isoalloxazine ring of the flavin contains several reactive dase has been proposed to occur via formation of a transient N5- centers or “hot spots” that play important roles in catalysis. In re- covalent intermediate [23]. In these examples, after the initial actions catalyzed by flavin monooxygenases, the activation of ox- attack of the substrate/analogs, formation of a N5-iminium ion or ygen involves a single electron transfer from the reduced flavin and decay of the flavin N5-adduct is coupled to an elimination step (e.g., recombination of the flavin semiquinone with the superoxide ion nitrite, chloride) (Scheme 2). In the N5-iminium ion, a carbon atom forming a covalent intermediate at the C4a-atom, the C4a- is activated for nucleophilic attack by a hydroxide ion (or other peroxyflavin (Scheme 1A) [13]. Formation of this intermediate is nucleophile) as part of the catalytic cycle (Scheme 2). Formation of essential for O-O bond cleavage and substrate oxygenation [14,15]. flavin N5-iminium ions in the methyl transfer steps of the reaction Similarly, C4a-thiol covalent intermediates are formed in gluta- catalyzed by flavin-dependent enzymes in thymidylate biosyn- thione reductase reactions and are involved in the mechanism of thesis and tRNA modifications have also been documented [24,25]. light activation by light-, oxygen-, or voltage-sensitive (LOV) do- Formation of prenylated FMN in decarboxylation reactions by mains (Scheme 1B) [16]. ferulic acid decarboxylase, has been proposed to form via N5- In addition to covalent intermediates at the C4a-position of the iminum ion intermediates [26]. More recently, formation of N5- isoalloxazine ring, covalent intermediates involving the flavin N5 oxoammonium ions, which are important for hydroxylation in have also been characterized. Reactivity of the N5-atom was shown enterocin biosynthesis and in dibenzothiophene degradation, have using model compounds and oxidized flavoenzymes reacting with been proposed [27,28]. These reactions result in reduction of the cabanions (Scheme 1B) [17e19]. Our group and others demon- flavin and oxidation of the substrate/analog. Thus, the flavin must strated the role of a covalent flavin N5 intermediate (Scheme 1A) in cycle back to the oxidized state for sustained catalysis. the isomerization of UDP-galactopyranose (UDP-Galp) to UDP- Formation of N5-adducts in non-redox reactions involving the galactofuranose (UDP-Galf) by UDP-galactopyranose mutase flavin in the oxidized or reduced forms have also recently been (UGM). The role of the reduced flavin in the non-redox reaction demonstrated. In the reaction of alkyl-dihydroxyacetone phosphate catalyzed by UGM is the focus of this review. We will discuss recent synthase (ADPS), the substrate attacks the N5-atom of the oxidized biochemical, structural, and computational data that suggest that flavin, and formation of the N5-iminum ion facilitates elimination the flavin cofactor in UGM plays multiple roles in catalysis. We will of the fatty acid. Formation of the N5-iminium ion activates the C1- focus on the mechanism of activation of the flavin-N5 for covalent atom, which is attacked by the fatty alcohol (e.g., Nu: in Scheme 2) catalysis, the role of the oxygen atom at the C4 positon (C4O) in [29,30]. In the ADPS reaction, the substrate functions as a nucleo- proton transfer and activation of flavin-sugar intermediates, and of phile and attacks the N5-atom of the flavin, which functions as an the 20-OH of the ribityl chain in stabilization of conformational electrophile. changes that modulate bending of the reduced flavin, which is In the reaction catalyzed by UGM (Scheme 3), the flavin is essential for the proton transfer function of the C4O. required to be in the reduced state and functions as a nucleophile. In this reaction, the substrate is not activated for N5-adduct

Scheme 1. Structure of the isoalloxazine ring highlighting hot spots for covalent catalysis. (A) Hot spots of the 2-electron reduced ﬂavin. Molecular oxygen is activated and form a covalent intermediate at the C4a position in monooxygenases. The N5-atom functions as a nucleophile attacking the C1-atom of UDP-Galp in UGM. (B) Hot spots of the oxidized ﬂavin. The N5-atom can function as an electrophile that is attacked by carbanions forming N5-iminium ions. Similarly, covalent intermediates at the C4a-position occur by attack of thiols. P. Sobrado, J.J. Tanner / Archives of Biochemistry and Biophysics 632 (2017) 59e65 61

Scheme 2. Nucleophilic attack of carbanions results in the formation of an N5-alkyl intermediate. Formation of the N5-iminium ion is coupled to an elimination step.

adduct was demonstrated in eUGMs [41] and the structures of the free enzyme and in complex with UDP-Galp in the oxidized and reduced state have been elucidated [42e45]. These structures revealed conformation changes in the active site flaps, which open in the absence and close in the presence of substrate. Other conformational changes that are coupled to the redox state of the flavin were also shown and included bending of the isoalloxazine ring and movement of several amino acids in the active site that are Scheme 3. The reaction catalyzed by UGM. predicted to place the sugar in proper orientation for flavin attack [43,46]. In addition, the conformational change of a loop (the his- formation; instead, priming of the flavin-N5 is required. In the tidine loop), moves a conserved His residue over 7 Å so that it can 0 sections that follow, we describe the mechanism that primes the hydrogen bond with the ribityl 2 -OH (Fig. 1A). Conformational flavin-N5 for nucleophilic attack and the role of the flavin-C4O in change of the histidine loop also places the carbonyl oxygen of a proton transfer, which is required to activate reaction conserved Gly residue in hydrogen bonding distance with the N5- intermediates. atom of the reduced flavin [42,43,46]. These interactions were described to be important to properly orient the reacting molecular orbital centered around the N5-atom and hindering the flavin from 3. Priming the flavin-N5 for reactivity in non-redox reactions reacting with molecular oxygen. The importance of the histidine loop was validated through biochemical studies of a variant of UGM Work on UGMs from bacterial sources by the Liu and Blanchard from Aspergillus fumigatus (AfUGM) in which the conserved His63 groups established that the reduced flavin is required for catalysis was mutated to Ala (AfUGMH63A). The mutant enzyme was shown and that the glycosidic bond was broken during turnover [31,32]. to be inactive, and the reduced AfUGMH63A was 100 times more Later, the Kiessling group demonstrated that a flavin-N5-covalent reactive with molecular oxygen than the wild-type enzyme [47]. intermediate was formed during turnover [33]. The structures of The flavoenzyme type II isopentenyl diphosphate isomerase bacterial UGMs (bUGMs) in various redox states revealed the basic (IDI-2) also requires reduced flavin to catalyze a redox neutral re- UGM fold and insight into substrate recognition [34e36]. However, action [48]. Analysis of the oxidized and reduced structures of they were less informative in explaining the various steps involved Sulfolobus shibatae IDI-2 revealed the presence of redox-mediated in the activation of the flavin for catalysis in the UGM reaction [35]. conformational changes in a flexible loop [49,50]. In the structure Crystal structures of eukaryotic UGMs (eUGMs) eventually pro- of the reduced enzyme, backbone carbonyl oxygen atoms of the vided insight into the mechanism of flavin activation, as described flexible loop form hydrogen bonds with the 20OH and N5-atom next. (Fig. 1B). These interactions are reminiscent of those observed be- We have been working with eUGM, which share low sequence tween the histidine loop and the reduced FAD in UGM. It is identity to bUGMs (~15%) and are, on average, ~100- amino acid reasonable to assume that these interactions in IDI-2 modulate the longer than bUGMs [37e40]. The presence of a covalent flavin-N5-

Fig. 1. Hydrogen bonds that prime the flavin N5 for reactivity. (A) Redox-mediated conformational changes of the histidine loop in AfUGM. Upon reduction of the FAD, the histidine loop rearranges to bring conserved Gly62 and His63 within hydrogen bonding distance of the FAD. (B) Structure of reduced IDI-2 (PDB 2ZRV) showing the hydrogen bonding of an active site loop with the FAD. These hydrogen bonds are reminiscent of those in reduced UGM. 62 P. Sobrado, J.J. Tanner / Archives of Biochemistry and Biophysics 632 (2017) 59e65 reaction of the N5-atom in the acid/base steps; however; this re- turnover suggests that the interactions involving Gly62 and His63 mains to be demonstrated. in the wild-type enzyme are essential for catalysis. The structure of the UGM adduct in combination with quantum 4. Activation of intermediates in catalysis: role of the flavin- mechanics/molecular mechanics (QM/MM) molecular dynamics C4O in proton transfer (MD) studies performed by Pierdominici-Sottile et al., provided a clear view of the important steps for catalysis in UGM and evidence fl The structure of AfUGMH63A complexed with the substrate for new functionalities for the avin cofactor [47,51]. The simula- UDP-Galp showed the presence of a C1-galactose-N5-FAD adduct tions indicate that after formation of the N5-galactose adduct, the (PDB 5HHF)(Fig. 2) [47]. This remains the only crystal structure next step is deprotonation of the N5-atom by the C4O (Fig. 3). The containing a covalent intermediate of UGM. Besides the new bond distance between the N5-H and the C4O in the reduced FAD is 2.4 Å fl formed between the sugar and the FAD-N5 and the absence of the (from ~2.7 Å in the oxidized form) due to bending of the avin. MD bond between the sugar and the UDP (Fig. 2A), differences from the simulations indicate that the bending is further increased in the E-S complex structure (PDB code 3UTH) were observed. These transition state, decreasing the distance between these two atoms include the lack of a hydrogen bond between His63 and the FAD 20- to 1.5 Å. This process is stabilized by interactions of the positively fl OH, shifting of the Galp O5 atom by 1.2 Å (Fig. 2B), and the charged His63 with the electron rich avin (Fig. 3). displacement of the carbonyl of Gly62, which now faces away from The next step in the reaction is opening of the sugar ring. This the N5-atom (Fig. 2C). The fact that the mutant enzyme is unable to step is coupled to the formation of the N5-iminum ion and is facilitated by protonation of Galp O5 atom. Bending of the flavin, which brings the FAD C4OH and the Galp O5 together for proton transfer, is also required in this step (Fig. 3). Without His63, the distance between the FAD C4OH and the Galp O5 is not decreased by bending of the flavin ring preventing the proton transfer step. Therefore, the reaction will stall at the FAD-Galp step, accounting for why this intermediate is observed in the AfUGMH63A structure [47]. Further support of this step comes from the observation that the Galp O5 atom is shifted 1.2 Å towards the FAD C4O in the FAD- Galp adduct structure (Fig. 2B). Formation of the FAD-Galp-iminium ion activates the Galp C1 for attack by the C4-OH to generate the furanose form of the sugar. Deprotonation of the sugar C4-OH prior to this step is facilitated by the FAD C4O atom. The proton now at the FAD C4O position is then transferred back to the FAD N5 atom (Fig. 3). The final step is the attack of the Galf C1 by UDP to form UDP-Galf, which yields the reduced flavin.

5. Summary and outlook

The chemical properties of the flavin cofactor in redox reactions are well-characterized. In some of these reactions, it has been shown that covalent flavin reactions occur, such as in the formation of C4a-(hydro)peroxyflavins where the semiquinone reacts with the superoxide ion. Formation of the N5-iminium ion in the reaction of DAAO and NAO with nitroalkanes occurs via attack of a carbanion intermediate. In these examples, the substrate is activated in the active site and reacts with the N5- or-C4-atoms, which can be considered as “hot spots” of the isoalloxazine ring. In the non-redox reaction catalyzed by UGM, the substrate is not activated for reaction with the flavin. Instead, redox-mediated conformational changes place the substrate in proper position and prime the flavin-N5 for nucleophilic attack. Interestingly, a similar conformation is observed in the “activation loop” in ID-II. Thus, conformational changes that prime the N5-atom are observed in non- redox reactions where the reduced flavin plays a role as a nucleophile or an acid/base. In addition to modulating the flavin reactivity, the protein scaffold (e.g., histidine loop in UGM) plays an essential role in the bending of the flavin ring. Bending of the flavin in the UGM reaction is essential for the proton transfer steps, which are required for advancement of the catalytic cycle (e.g., ring opening) and refor- Fig. 2. Structure of a covalent intermediate bound to AfUGMH63A (PDB 5HHF). (A) fl Structure of the FAD-Galp intermediate with severed UDP. Reduced FAD is colored gray. mation of the reduced avin. These proton transfer steps involve Severed UDP-Galp is colored pink. (B) Comparison of the covalent intermediate in the FAD C4O atom (Fig. 3). Studies on UGM show that the FAD C4O- AfUGMH63A (white) and the noncovalent E-S complex (cyan, PDB code 3UTH). For- atom plays a role in acid/base reactions and should also be mation of the N5-C1 bond draws the Galp O5 closer to the flavin O4. (C) Comparison of considered as a reactive center or hot spot. The role of the flavin in the loops of AfUGMH63A (white) and the E-S complex (cyan). Black and yellow dashes the acid/base reaction is also observed in IDI-2, however, the N5- indicate hydrogen bonds in AfUGMH63A and the E-S complex, respectively. Note that Gly62 fails to hydrogen bond to the FAD N5 atom in the covalent complex, suggesting atom is predicted to be the main player in that system [52]. 0 that this interaction in the wild-type enzyme is essential for turnover. Interaction of the imidazole group of H63 in AfUGM with the 2 - P. Sobrado, J.J. Tanner / Archives of Biochemistry and Biophysics 632 (2017) 59e65 63

Fig. 3. (A) Diagram showing the major steps in the chemical mechanism. UGM with reduced flavin binds to UDP-Galp (1) and a covalent flavin-galactose adduct is formed via the direct attack of the FAD-N5 to the Galp-C1 atom (2). This step leads to cleavage of the glycosidic bond. Bending of the flavin permits the transfer of the proton from the FAD N5 (shown in red) to the C4O (3). This proton is next transferred to the Galp C5O-atom, facilitating the opening of the sugar ring and formation of the flavin iminium ion (4). The FAD C4O is predicted to accept the proton from the Galp C4-OH (shown in blue) during ring contraction (5). The next step is the transfer of the proton from the FAD C4O to the FAD N5 (6). The final product is formed by direct attack of UDP to the FAD-Galf adduct (7) and release of UDP-Galf from the active site completes the catalytic cycle (8). (B) Structure of the transition state for the transfer of the proton from the FAD N5 to the C4O (Step 3). (C) Structure of the transition state for the protonation of the Galp5O by the FAD4O (Step 4). The coordinates were provided by Prof. Pierdominici [51].Thefirst number for the amino acids are those for AfUGM and the second for TcUGM.

OH of the ribityl chain is important in establishing the various in- which alter the hydrogen bonding pattern of the 20-OH [54e57]. teractions of the His loop with the flavin ring. Other evidence of the The new interactions are thought to be important for stabilizing the 20-OH of the ribityl chain playing a role in catalysis has been reduced FAD and broadcasting the flavin redox state to the rest of observed in NAO where it interacts with and facilitates elimination the enzyme. Thus, the 20-OH of the ribityl chain can be considered of the nitrate group [21]. Hydrogen bonding between the ribityl 20- as a flavin hot spot. OH atom and the carbonyl oxygen of the substrate in medium chain In summary, the flavin cofactor in UGM plays a role in the for- acyl-CoA dehydrogenase have been shown to play an important mation of N5-adducts and in intra-flavin and flavin-substrate pro- role in the activation of C-H bond during hydride transfer [53].In ton transfer steps (between the N5 and C4O atoms and with the the bifunctional enzyme proline utilization A, reduction of the FAD Galp O5). Priming of the N5-atom and bending of the flavin during induces dramatic conformational changes of the ribityl chain, proton transfer requires redox-mediated conformations changes, 64 P. Sobrado, J.J. Tanner / Archives of Biochemistry and Biophysics 632 (2017) 59e65

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